Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows

J. Dairy Sci. 98:1–13 http://dx.doi.org/10.3168/jds.2014-9294 © American Dairy Science Association®, 2015.

Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows J. Oh,* F. Giallongo,* T. Frederick,* J. Pate,* S. Walusimbi,* R. J. Elias,† E. H. Wall,‡ D. Bravo,§ and A. N. Hristov*1 *Department of Animal Science, and †Department of Food Science, The Pennsylvania State University, University Park 16802 ‡Pancosma S.A., CH-1218 Geneva, Switzerland §InVivo Animal Nutrition & Health, Talhouët, 56250 Saint-Nolff, France

ABSTRACT

energy-corrected milk yield was quadratically increased by CAP, possibly as a result of enhanced mobilization of body fat reserves. In addition, CAP increased neutrophil activity and immune cells related to acute phase immune response. Key words: capsicum oleoresin, milk production, immune response, dairy cow

This study investigated the effect of Capsicum oleoresin in granular form (CAP) on nutrient digestibility, immune responses, oxidative stress markers, blood chemistry, rumen fermentation, rumen bacterial populations, and productivity of lactating dairy cows. Eight multiparous Holstein cows, including 3 ruminally cannulated, were used in a replicated 4 × 4 Latin square design experiment. Experimental periods were 25 d in duration, including a 14-d adaptation and an 11-d data collection and sampling period. Treatments included control (no CAP) and daily supplementation of 250, 500, or 1,000 mg of CAP/cow. Dry matter intake was not affected by CAP (average 27.0 ± 0.64 kg/d), but milk yield tended to quadratically increase with CAP supplementation (50.3 to 51.9 ± 0.86 kg/d). Capsicum oleoresin quadratically increased energy-corrected milk yield, but had no effect on milk fat concentration. Rumen fermentation variables, apparent total-tract digestibility of nutrients, and N excretion in feces and urine were not affected by CAP. Blood serum β-hydroxybutyrate was quadratically increased by CAP, whereas the concentration of nonesterified fatty acids was similar among treatments. Rumen populations of Bacteroidales, Prevotella, and Roseburia decreased and Butyrivibrio increased quadratically with CAP supplementation. T cell phenotypes were not affected by treatment. Mean fluorescence intensity for phagocytic activity of neutrophils tended to be quadratically increased by CAP. Numbers of neutrophils and eosinophils and the ratio of neutrophils to lymphocytes in peripheral blood linearly increased with increasing CAP. Oxidative stress markers were not affected by CAP. Overall, in the conditions of this experiment, CAP did not affect feed intake, rumen fermentation, nutrient digestibility, T cell phenotypes, and oxidative stress markers. However,

INTRODUCTION

Capsicum is a genus of flowering plants containing capsaicinoids as its active compounds. Original interest in Capsicum as a feed additive in beef and dairy production systems was based on its potential as modifier of rumen fermentation and its pungency to increase feed intake (Calsamiglia et al., 2007; Rodríguez-Prado et al., 2012). Recent findings suggested, however, that the improvement in animal performance may be due to a host response (physiological and immunological) rather than direct antimicrobial or sensory effects. For example, Capsicum oleoresin or its mixture with other plant extracts have reduced oxidative stress (Karadas et al., 2014), prevented disease symptoms (Lee et al., 2011, 2013; Liu et al., 2012), and improved gut health during normal or disease conditions (Liu et al., 2014a,b) in both poultry and swine. In addition, studies using rats showed that capsaicinoids, active compounds in Capsicum, had modulatory effects on immune cells such as neutrophils and T cells (Franco-Penteado et al., 2006; Takano et al., 2007), and decreased oxidative stress markers and induced negative energy balance (Yoshioka et al., 2000; Abdel-Salam et al., 2012). With respect to ruminants, Oh et al., (2013) reported that a short-term (9-d) abomasal infusion of Capsicum oleoresin increased lymphocyte proportion and CD4+ T cells and did not affect oxidative stress markers in blood of dairy cows. However, it was unknown if such host-based physiological effects of Capsicum would be observed when supplementation is through the feed versus postruminal delivery.

Received December 30, 2014. Accepted June 5, 2015. 1 Corresponding author: [email protected]

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Based on the existing data, we hypothesized that dietary supplementation of Capsicum oleoresin in unprotected granular form (CAP) could potentially modify rumen fermentation, facilitate immune response, and reduce oxidative stress in dairy cows. The CAP product could also stimulate feed intake and animal productivity. Thus, the objective of the experiment was to investigate the physiological effects of CAP in relation to feed intake, rumen function, blood chemistry, hematology, immune responses, oxidative stress markers, and productivity of lactating dairy cows. MATERIALS AND METHODS Animals and Treatments

The Pennsylvania State University Animal Care and Use committee approved all procedures in this experiment. The design of the experiment was a replicated 4 × 4 Latin square with 8 multiparous Holstein cows averaging 590 ± 32.6 kg of BW, 50 ± 9.6 DIM, and 52 ± 2.4 kg/d milk yield at the beginning of the experiment. Three cows were fitted with soft plastic rumen cannulas (10 cm internal diameter; Bar Diamond Inc., Parma, ID) and constituted 1 incomplete square of the experiment. Experimental periods were 25 d, including a 14-d adaptation and an 11-d data collection period. Treatments were control (0 mg/d of CAP per cow) and 3 application rates of CAP: 250 (C250), 500 (C500), and 1,000 mg/d per cow (C1000). The CAP product used in the experiment was CapsXL (X60–7035; 20% Capsicum oleoresin; 1.2% capsaicinoids; Pancosma S. A., Geneva, Switzerland). The product was top-dressed daily mixed with a small amount of TMR during feeding. Cows were housed in a tiestall barn, fed once daily at approximately 0800 h, and had free access to fresh water. The basal diet (Table 1) was fed ad libitum as a TMR, targeting approximately 10% refusals. The diet was formulated to meet or exceed the nutrient requirements of a lactating Holstein cow yielding 50 kg of milk/d with 3.80% fat and 3.20% true protein at 27 kg/d of DMI and 650 kg of BW (NRC, 2001). Cows were milked twice daily at 0500 and 1700 h and treated with recombinant bST (rbST; 500 mg, i.m., Posilac; Elanco Co., Greenfield, IN) on d 1 and 13 of each experimental period. Sampling and Analyses

Feed intake was measured daily, TMR samples were collected twice weekly, and individual ingredients of forage and concentrate were sampled once weekly. Composite samples of the TMR, forages, and concentrate feeds were processed and analyzed for OM, CP, Journal of Dairy Science Vol. 98 No. 9, 2015

NDF, ADF, Ca, P, indigestible NDF (iNDF), and NEL as described in Oh et al. (2013). Milk production of the cows was recorded daily and samples for milk composition were collected on d 20 and 24 of each experimental period from evening and

Table 1. Ingredient and chemical composition of the diet fed during the experiment Item Ingredient, % of diet DM   Corn silage1   Grass hay2   Cottonseed, hulls   Corn grain, ground   Candy by-product meal3   Soybean seeds, whole heated4   Canola meal5  Molasses6   Vitamin and mineral premix7  SoyPLUS8  Optigen9 Composition,10 % of DM   (unless otherwise noted)  CP10  RDP11  RUP11  NDF10  ADF10  NEL, Mcal/kg10  Ca10  P10  NFC11   Average NEL balance,12 Mcal/d   Average MP balance,12 g/d 1

Measurement  

44.0 7.0 5.5 9.5 1.8 8.0 12.0 5.4 3.0 3.4 0.4  

15.8 10.2 7.0 30.7 18.8 1.58 0.70 0.40 43.2 0.2, −0.9, −0.9, and 0.4 −140, −137, −163, and −57

Corn silage was 46.5% DM and contained (DM basis) 8.5% CP, 32.1% NDF, and 41.4% starch. 2 Grass hay was 90.7% DM and contained (DM basis) 7.0% CP and 73.8% NDF. 3 Candy by-product meal (Graybill Processing, Elizabethtown, PA) contained (DM basis) 16.6% CP and 27.8% NDF. 4 Soybean seeds contained (DM basis) 40.0% CP. 5 Canola meal contained (DM basis) 41.8% CP. 6 Molasses (Westway Feed Products, Tomball, TX) contained (DM basis) 3.9% CP and 66% total sugar. 7 The premix (Cargill Animal Nutrition, Cargill Inc., Roaring Spring, PA) contained (%, as-is basis) trace mineral mix, 0.86; MgO (56% Mg), 8.0; NaCl, 6.4; vitamins A, D, and E premix (Cargill Animal Nutrition), 0.48; limestone, 37.2; selenium premix (Cargill Animal Nutrition), 0.07; and dry corn distillers grains with solubles, 46.7; Ca, 14.1%; P, 0.39%; Mg, 4.59%; K, 0.44%; S, 0.39%; Se, 6.91 mg/kg; Cu, 362 mg/kg; Zn, 1,085 mg/kg; Fe, 186 mg/kg, vitamin A, 276,717 IU/ kg; vitamin D, 75,000 IU/kg; and vitamin E, 1,983 IU/kg. 8 SoyPLUS (West Central Cooperative, Ralston, IA) contained (DM basis) 46.6% CP. 9 Optigen is a slow-release urea (Alltech Inc., Nicholasville, KY). 10 Values ​​calculated using the chemical analysis (Cumberland Valley Analytical Services Inc., Maugansville, MD) of the ingredients of the diet. 11 Estimated by NRC (2001). 12 Estimated based on NRC (2001) using actual DMI, milk yield, milk composition, and BW of the cows throughout the experiment (Control, C250, C500, and C1000, respectively).

CAPSICUM OLEORESIN IN DAIRY COWS

morning milkings, preserved with 2-bromo-2-nitropropane-1,3 diol, and submitted to Dairy One laboratory for analysis (Pennsylvania DHIA, University Park, PA). Milk was analyzed for fat, true protein, lactose, and MUN using infrared spectroscopy (MilkoScan 4000; Foss Electric, Hillerød, Denmark). Another aliquot was collected in tube containing no preservative, kept at −20°C, and analyzed for milk FA composition as described in Hristov et al. (2010). Samples of whole ruminal contents were collected from the cannulated cows on d 24 and 25 of each experimental period at 2, 4, and 6 h after feeding, processed as described elsewhere (Hristov et al., 2011), and analyzed for pH (59000–60 pH Tester, Cole-Parmer Instrument Company, Vernon Hills, IL), VFA (Yang and Varga, 1989), and ammonia concentration (Chaney and Marbach, 1962). Aliquots of whole ruminal contents were composited (on an equal wet weight basis), per cow and period, and stored frozen at −80°C for bacterial population analysis. The 16S rRNA gene V4 variable region PCR primers 515/806 (Caporaso et al., 2011) were used in a single-step 30-cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, Germantown, MD) under the following conditions: 94°C for 3 min, followed by 28 cycles (5 cycles used on PCR products) of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was completed. Sequencing was performed at Molecular Research DNA (www.mrdnalab.com; Shallowater, TX) on an Ion Torrent PGM (Life Technologies, Carlsbad, CA) following the manufacturer’s guidelines. Sequence data were processed using a proprietary analysis pipeline (Molecular Research DNA). In summary, sequences were depleted of barcodes and primers, then sequences <150 bp were removed, and sequences with ambiguous base calls and with homopolymer runs exceeding 6 bp were also removed. Sequences were denoised, operational taxonomic units generated, and chimeras removed. Operational taxonomic units were defined by clustering at 3% divergence (97% similarity). Final operational taxonomic units were taxonomically classified using BLASTn against a curated database derived from GreenGenes, RDPII, and NCBI (www.ncbi.nlm. nih.gov; DeSantis et al., 2006, http://rdp.cme.msu.edu; accessed Dec. 24, 2014). Spot fecal and urine samples were collected by stimulating defecation or from the rectum and by massaging the vulva, respectively, at 1000, 1600, and 2200 h on d 22; 0400, 1300, and 1900 h on d 23; and 0100 and 0700 h on d 24 of each experimental period. Fecal and urine samples were collected, processed and analyzed for OM, CP, NDF, and ADF (fecal samples) and total N, urinary urea N, creatinine, and the purine derivatives (PD) allantoin and uric acid (urine samples) as

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described in Oh et al. (2013). Daily volume of excreted urine was estimated based on urinary creatinine concentration, assuming a creatinine excretion rate of 29 mg/kg of BW (determined based on total urine collection samples from Hristov et al., 2011). Estimated urine output was used to calculate daily total N and urinary urea N excretions. Apparent total-tract digestibility of nutrients was estimated using iNDF as an intrinsic digestibility marker (Schneider and Flatt, 1975). Fecal and TMR samples were analyzed for iNDF according to Huhtanen et al. (1994), with the exception that 25µm pore size Ankom filter bags (Ankom Technology, Macedon, NY) were used for the rumen incubation. Blood samples were collected from the coccygeal vein or artery at 2 and 4 h after feeding on d 22 of each period for hematology analyses. Samples (approximately 10 mL) were collected into vacuumed tubes containing EDTA (BD Biosciences, Franklin Lakes, NJ), kept refrigerated (4°C), and analyzed the same day. The analysis included red blood cell count, hemoglobin, hematocrit, platelet count, mean platelet volume, and total white blood cell count, including total count for neutrophils, eosinophils, lymphocytes, monocytes, and basophils using an automated hematology analyzer (HemaVet; Drew Scientific, Oxford, CT). A separate set of blood samples was collected into vacuumed tubes containing silica clot activator (SST Tube; BD Biosciences) at 2 and 4 h after feeding on d 22 and 23 of each experimental period. Blood serum was separated (after clotting) through centrifugation at 3,000 × g at room temperature for 15 min, composited on an equal volume basis per cow, period, and sampling time point and stored at −20°C. Serum was analyzed for albumin, amylase, BUN, Ca, cholesterol, Cl, creatinine, globulin, glucose, K, Na, P, and total protein (Idexx VetTest and VetLyte Chemistry and Electrolyte Analyzers, Idexx Laboratories Inc., Westbrook, ME). Serum samples were also analyzed for BHBA and NEFA using biochemistry analyzer (Cobas 6000; Roche, Berlin, Germany) and for insulin using RIA (Coat-a-count insulin kit TKIN5; Siemens Healthcare Diagnostics, Los Angeles, CA). A third set of blood samples was collected into vacuumed tubes containing EDTA (BD Biosciences) at 2 and 4 h after feeding on d 22 and 23 of each experimental period for oxidative stress markers. Plasma was obtained by centrifugation at 1,500 × g at 4°C for 10 min, composited on an equal volume basis per cow, period, and sampling time point, and stored frozen at −80°C. Samples were analyzed for thiobarbituric acid reactive substances using colorimetric assay kits (Cayman Chemical, Ann Arbor, MI) and 8-isoprostane using ELISA kits (Cayman Chemical) and for oxygen radical absorbance capacity, as described elsewhere (Cao and Prior, 1999). Journal of Dairy Science Vol. 98 No. 9, 2015

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Whole-blood (approximately 50 mL) samples were collected via tail vein or artery before feeding from 1 experimental square (4 cows) on d 24 and from the other square (4 cows) on d 25 of each period for T cell phenotype analysis. Whole blood was transferred to borosilicate glass tubes and centrifuged at 1,500 × g at 4°C for 10 min to obtain a lymphocyte-rich white blood cell layer. Peripheral blood mononuclear cells were isolated by centrifugation at 650 × g at 25°C for 30 min over Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Piscataway, NJ). A flow cytometer (GuavaEasyCyte Plus; Millipore, Billerica, MA) was used for counting and analysis of T lymphocytes. Peripheral blood mononuclear cells (1 × 106) were directly labeled with fluorconjugated primary antibodies against T lymphocytesurface antigens following a protocol described by Poole and Pate (2012). The antibodies used were against cluster of differentiation antigen (CD) 4 (MCA1653F; AbD Serotech, Raleigh, NC), CD25α (CACT116A; VMRD, Pullman, WA), CD8α (MCA837F; AbD Serotech), and δ T cell receptor (δTCR; CACT61A, VMRD). The primary antibodies for CD25α and δTCR were conjugated to phycoerythrin using IgG1 and IgM goat anti-mouse antibodies [IgG1 (STAR132P) and IgM (102009), respectively; AbD Serotech] following the manufacturers guidelines. The following antibodies were used as controls: IgG2a negative control (MCA929F; AbD Serotech), IgG1 negative control (MCA928PE and MCA928F; AbD Serotech), and IgM negative control (MCA692; AbD Serotech). Samples were analyzed in duplicate, CD4 was paired with CD25α and CD8 was paired with δTCR. Flow cytometer (GuavaEasyCyte Plus; Millipore) was used to determine the proportion of single- and dual-stained cells. The phagocytic capacity of neutrophils was measured by a flow cytometric assay (Smits et al., 1997). Killed bacteria, Streptococcus uberis, were provided by the Animal Diagnostic Laboratory at the Pennsylvania State University, suspended in 1 mL of PBS, and labeled with propidium iodide (PI) for 60 min at room temperature. The PI-labeled bacteria were washed 3 times with PBS to remove excess PI, suspended in RPMI1640 medium (Gibco Laboratories, Grand Island, NY), and counted using a flow cytometer (GuavaEasyCyte Plus, Millipore). Aliquots were stored at −80°C and thawed immediately before use. Whole blood (10 mL) was collected from the coccygeal vein or artery into heparinized-vacutainers (BD Biosciences) on d 23 of each experimental period. The blood was centrifuged at 4°C and 1,500 × g for 10 min, the supernatant and buffy coat layers were aspirated and discarded, and the packed red blood cells were lysed by the addition of 2 volumes of red blood cell lysis buffer (0.14 M NH4Cl, 10 mM KHCO3). Lysis was stopped by adding a volJournal of Dairy Science Vol. 98 No. 9, 2015

ume of Hanks’ balanced salt solution after 1 min. The lysate was centrifuged at 400 × g at 4°C for 10 min with no brake, washed with Hanks’ balanced salt solution once at 300 × g at 4°C for 10 min, and finally suspended in RPMI-1640 medium. Total neutrophils were counted by flow cytometer (GuavaEasyCyte Plus, Millipore). Bacteria labeled with PI were added into 96-well plates at a ratio of the bacteria to neutrophils of 15:1 (bacteria:neutrophils = 15 × 105:1 × 105). After 20 min of incubation at room temperature, the plates were read on a flow cytometer (GuavaEasyCyte Plus, Millipore) and the proportion of neutrophils that had phagocytized bacteria was determined. Also, histogram analysis for mean fluorescence intensity of PI was conducted to estimate mean phagocytic activity of total gated neutrophils, which indicated mean number of engulfed bacteria per neutrophil (Silvestre et al., 2011). The assay was run in duplicate. Statistical Analysis

All data were analyzed using the MIXED procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC). Milk yield, DMI, and estimated feed efficiency data for the last 11 d and milk composition data for the 2 sampling days of each experimental period were averaged and the average values were used in the statistical analysis. The averaged milk yield, DMI, and milk composition data were used to calculate yields of milk fat, protein, lactose, 4.0% FCM, and ECM. Nutrient intake, digestibility, rumen microbial population, urinary and fecal N excretions, milk composition, hematology, blood chemistry, BHBA, NEFA, T cell phenotypes, and neutrophil phagocytosis data were analyzed by ANOVA Latin square. The model used was as follows: Yijkl = μ + Si + C(S)ij + Pk + Tl + eijkl, where Yijkl is the dependent variable, μ is the overall mean, Si is the square, C(S)ij is the cow within square, Pk is the kth period, and Tl is the lth treatment with the error term eijkl. Square and cow within square were random effects and all others were fixed. Rumen fermentation data (pH and ammonia and VFA concentrations), DMI, milk yield, SCC, and feed efficiency data were analyzed as repeated measures assuming an AR(1) covariance structure. The model used was as follows: Yijklm = μ + Si + C(S)ij + Pk + Tl + Dm + TDlm + eijklm,

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CAPSICUM OLEORESIN IN DAIRY COWS

where Yijklm is the dependent variable, μ is the overall mean, Si is the square, C(S)ij is the cow within square, Pk is the kth period, Tl is the lth treatment, Dm is the time effect, and TDlm is the treatment × time of sampling interaction, with the error term eijklm. Square and cow within square were random effects and all others were fixed. Data were tested for normality using the UNIVARIATE procedure of SAS. Log-transformed data were analyzed when the W statistic of the Shapiro-Wilk test was less than 0.05. Orthogonal contrasts were used to evaluate CAP treatments versus control, linear, and quadratic effects of CAP supplementation. Statistical differences were considered significant at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. RESULTS

The basal diet used in this experiment was formulated to meet NEL and MP requirements of cows milking 50 kg/d. Due to higher than expected milk yield and BW deposition during the experiment, however, the diet provided NEL slightly below NRC (2001) requirements (−2.0 and −2.1% for C250 and C500, respectively) and MP from −1.8 to −5.0% below the requirements (Table

1). Cows gained, on average, 34 ± 10.3 kg of BW during the experiment (P = 0.26). Dry matter intake was not affected by CAP supplementations (Table 2). Milk yield tended to quadratically increase (P = 0.09) with CAP. Cows in this experiment were experiencing milk fat depression. Despite numerical differences, milk fat concentration was not affected by treatments, but milk fat yield was higher (P = 0.05) for CAP than the control and tended to be quadratically increased (P = 0.09) by CAP supplementation. Compared with the control, 4% FCM and ECM were increased (P = 0.04 and P = 0.06, respectively) by CAP and quadratically increased (P = 0.04) with increasing CAP supplementation rate. No effect of CAP supplementation on milk true protein content and yield was observed. Concentration of lactose linearly decreased (P < 0.01), but concentrations of TS and MUN were not affected by CAP. Milk NEL increased quadratically (P = 0.05) with CAP supplementation. As dietary NEL intake remained constant, milk NEL efficiency was higher (P = 0.05) for CAP than the control. Treatment did not affect milk SCC and BW of the cows. The milk FA data from this experiment are shown in Table 3. Saturated FA in milk were not affected by CAP, except 18:0 and 20:0. Concentration of 18:0 tend-

Table 2. Effect of dietary Capsicum oleoresin (CAP) on DMI, milk yield and composition, and BW in dairy cows Treatment1

P-value3

Item

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

DMI, kg/d Milk yield, kg/d Feed efficiency4, kg/kg Milk fat, % Yield, kg/d 4% FCM, kg/d ECM,5 kg/d Milk true protein, % Yield, kg/d Milk lactose, % Yield, kg/d TS, % MUN, mg/100 mL Milk NEL,6 Mcal/d Dietary NEL intake, Mcal/d Milk NEL efficiency,7% SCC, ×103 cells/mL BW, kg

27.0 50.5 1.90 2.89 1.46 42.3 46.6 3.01 1.53 4.86 2.46 11.8 16.6 32.0 42.7 74.9 172 623

27.5 51.9 1.93 3.12 1.62 45.4 49.5 3.00 1.56 4.82 2.50 12.0 16.1 33.9 43.1 79.0 133 621

27.0 51.5 2.02 3.03 1.55 44.2 48.3 2.98 1.54 4.78 2.49 11.8 16.7 33.0 42.4 77.8 77.2 614

26.5 50.3 1.96 3.07 1.56 43.4 47.3 2.98 1.50 4.78 2.41 11.8 15.8 32.3 41.7 77.6 138 614

0.64 0.86 0.047 0.010 0.054 1.01 1.02 0.036 0.038 0.016 0.046 0.13 0.69 0.78 1.02 1.86 86.5 10.3

0.75 0.37 0.16 0.12 0.05 0.04 0.06 0.43 0.85 0.01 0.96 0.48 0.34 0.09 0.76 0.05 0.40 0.26

0.27 0.54 0.27 0.36 0.60 0.79 0.98 0.34 0.30 <0.01 0.29 0.99 0.21 0.83 0.24 0.30 0.67 0.16

0.84 0.09 0.25 0.33 0.09 0.04 0.04 0.73 0.26 0.13 0.29 0.65 0.63 0.05 0.65 0.13 0.33 0.57

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Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. Highest SEM shown; n = 359 for DMI and dietary NEL intake, n = 353 for milk yield and feed efficiency, n = 339 for BW, n = 32 for all other variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 4 Milk yield ÷ DMI. 5 ECM (kg/d) = kg of milk × [(38.3 × % fat × 10 + 24.2 × % true protein × 10 + 16.54 × % lactose × 10 + 20.7) ÷ 3,140] (Sjaunja et al., 1990). 6 Milk NEL (Mcal/d) = kg of milk × (0.0929 × % fat + 0.0563 × % true protein + 0.0395 × % lactose) (NRC, 2001). 7 Milk NEL ÷ NEL intake × 100. 2

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ed to linearly increase (P = 0.09) and that of 20:0 was linearly increased (P = 0.03) by CAP compared with the control. Milk 18:2 cis-9,cis-12, and 18:1 trans-11 tended to linearly decrease (P = 0.10 and 0.07, respectively), whereas other 18:1 trans and cis FA were not affected by CAP. Supplementation with CAP linearly increased (P = 0.04) concentration of 18:3. Compared with the control, concentration of CLA cis-9,trans-11 was decreased (P = 0.03) by CAP. The sum of saturated FA, total trans FA, and MUFA were not affected by CAP. Concentration of PUFA was linearly decreased (P = 0.05) by CAP. Rumen ammonia and VFA concentrations were not affected by CAP supplementation (Table 4). Rumen pH tended to decrease (P = 0.08) with CAP compared with the control. Intake and apparent total-tract digestibility of dietary nutrients were not affected by treatment (Table 5). Supplementation with CAP also did not affect N excretion in feces or urine, urine volume, urinary urea N excretion, total excreta N, milk N secretion, and urinary PD excretion (Table 6). Relative to the control, plasma BHBA concentration was quadratically increased (P = 0.02) by CAP, but no

effect of CAP on NEFA and insulin concentrations was noted (Table 7). Plasma BHBA concentration in our experiment was below the level for subclinical ketosis (1,200 to 2,900 µM; Oetzel, 2004). Supplementation of the diet with CAP increased (P = 0.02) BUN compared with the control. Concentrations of glucose, creatinine, total protein, albumin, globulin, amylase, Na, and Cl in blood plasma were not affected by CAP except cholesterol that tended to quadratically increase (P = 0.07). Plasma K concentration linearly decreased (P < 0.01) with CAP supplementation. As revealed by the sequencing results, the predominant bacterial orders in whole ruminal contents were Clostridiales and Bacteroidales (Table 8). Bacteroidales decreased quadratically (P = 0.03) and Bifidobacteriales tended to be linearly increased (P = 0.10) by CAP. Relative to the control, Synergistales increased quadratically (P = 0.03) and Spirochaetales tended to be quadratically increased (P = 0.09) by CAP. The relative abundance of Ruminococcaceae, which was the most predominant genus in the rumen, was not affected by treatment (Table 9). Prevotella and Roseburia were quardratically decreased (P ≤ 0.04) and the proportion

Table 3. Effect of dietary Capsicum oleoresin (CAP) on milk FA in dairy cows (% of milk fat) Treatment1 Item 4:0 6:0 8:0 10:0 12:0 14:0 14:1 15:0 16:0 16:1 17:0 18:0 18:1 trans-6–8 18:1 trans-9 18:1 trans-10 18:1 trans-11 18:1 trans-12 18:1 cis-9 18:1 cis-11 18:2 cis-9,cis-12 18:3 20:0 CLA cis-9,trans-11 CLA trans-10,cis-12 Σ unidentified Σ SFA Σ trans FA Σ MUFA Σ PUFA

P-value3

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

3.31 1.76 1.03 2.42 2.85 9.13 1.18 0.95 22.7 1.69 0.42 9.40 0.56 0.45 2.32 1.33 0.61 24.9 1.91 4.55 0.43 0.10 0.61 0.01 5.29 54.1 5.33 35.0 5.60

3.38 1.85 1.09 2.57 2.95 9.26 1.05 0.96 23.2 1.52 0.43 9.54 0.57 0.44 2.45 1.26 0.58 24.3 1.87 4.49 0.43 0.10 0.58 0.01 5.26 55.3 5.16 33.9 5.51

3.54 1.84 1.06 2.45 2.80 8.90 0.99 0.86 22.9 1.50 0.42 9.85 0.57 0.42 2.19 1.12 0.54 25.5 1.85 4.42 0.42 0.10 0.54 0.01 5.11 54.8 4.90 34.7 5.40

3.47 1.84 1.06 2.47 2.86 9.06 1.07 0.88 23.1 1.58 0.42 10.0 0.58 0.46 2.53 1.09 0.58 24.5 1.85 4.34 0.41 0.11 0.51 0.01 5.12 55.3 5.31 34.3 5.27

0.211 0.105 0.047 0.098 0.083 0.310 0.108 0.077 1.69 0.116 0.027 0.378 0.035 0.024 0.403 0.181 0.037 1.84 0.062 0.099 0.009 0.004 0.077 0.002 0.151 2.29 0.337 2.00 0.173

0.62 0.73 0.82 0.97 0.99 0.77 0.79 0.20 0.45 0.74 0.57 0.21 0.86 0.58 0.48 0.78 0.94 0.12 0.11 0.81 0.22 0.21 0.03 0.20 0.07 0.57 0.93 0.30 0.62

0.24 0.21 0.31 0.38 0.61 0.52 0.30 0.41 0.69 0.30 0.95 0.09 0.86 0.85 0.82 0.07 0.39 0.87 0.25 0.10 0.04 0.03 0.08 0.73 0.11 0.46 0.52 0.73 0.05

0.19 0.30 0.50 0.61 0.90 0.62 0.25 1.00 0.70 0.36 0.57 0.77 0.56 0.18 0.19 0.50 0.15 0.54 0.46 0.84 0.73 0.88 0.76 0.60 0.44 0.67 0.25 0.72 0.80

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. n = 32 for all variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 2

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Table 4. Effect of dietary Capsicum oleoresin (CAP) on rumen fermentation in dairy cows Treatment1

P-value3

Item

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

pH Ammonia, mM Total VFA, mM Total VFA, %  Acetate  Propionate  Butyrate  Isobutyrate  Valerate  Isovalerate  Acetate:propionate

5.86 2.22 140   50.8 31.1 12.6 0.64 3.35 1.47 1.66

5.78 2.22 139   51.3 30.4 12.9 0.63 3.37 1.56 1.70

5.76 2.21 147   50.5 31.4 12.3 0.61 3.56 1.56 1.62

5.78 2.03 138   49.8 31.9 12.5 0.63 3.48 1.54 1.60

0.075 0.284 6.3   0.81 1.16 0.58 0.016 0.474 0.042 0.083

0.08 0.70 0.86   0.59 0.85 0.93 0.42 0.52 0.13 0.71

0.22 0.32 0.88   0.10 0.34 0.75 0.77 0.49 0.39 0.22

0.23 0.60 0.32   0.53 0.74 0.88 0.17 0.58 0.23 0.76

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. Highest SEM shown; n = 71 for VFA, n = 72 for all other variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 2

of Butyrivibrio was increased quadratically (P < 0.01) by CAP supplementation. Capsicum quadratically increased (P ≤ 0.04) the abundance of Fecalibacterium, Syntrophococcus, and Dorea. T cell phenotypes were not affected by CAP supplementation (Table 10). Although CAP did not affect numbers of neutrophils positive for phagocytosis, mean fluorescence intensity tended to be quadratically increased (P = 0.08). Supplementation with CAP linearly increased total white blood cells, neutrophils, and eosinophils (P ≤ 0.04; Table 11). The proportion of lymphocytes in total white blood cells decreased linearly (P < 0.01) and that of neutrophils increased linearly (P < 0.01) with increasing CAP supplementation; therefore, the ratio of neutrophils to lymphocytes was linearly increased (P < 0.01) by CAP. Treatment did

not affect concentrations of monocytes and basophils in blood. Red blood cells and hemoglobin concentration quadratically increased (P ≤ 0.04) and platelets linearly decreased (P = 0.04) with CAP, even though mean platelet volume was not affected. Hematocrit percentage tended to be increased (P = 0.07) by CAP. Oxidative stress markers were not affected by CAP supplementation (Table 12). DISCUSSION

Supplementation on the diet with Capsicum oleoresin increased feed intake in beef cattle (Cardozo et al., 2006; Rodríguez-Prado et al., 2012). In these experiments, the pungent property of Capsicum led to high consumption of water followed by increased feed intake. However,

Table 5. Effect of dietary Capsicum oleoresin (CAP) on intake and apparent total-tract digestibility of dietary nutrients in dairy cows Treatment1 Item Intake, kg/d  DM4  OM  CP  NDF  ADF  Starch Apparent digestibility, %  DM  OM  CP  NDF  ADF  Starch

Control  

27.9 26.3 4.39 9.32 6.52 6.11   66.3 67.3 65.7 42.8 38.1 95.8

C250  

28.0 26.4 4.42 9.37 6.45 6.13   66.1 67.0 65.7 41.2 38.2 95.4

P-value3

C500  

27.4 25.8 4.31 9.15 6.32 6.00   66.9 67.9 67.2 42.5 37.7 96.3

SEM2

C1000  

27.0 25.4 4.24 9.02 6.20 5.91   67.2 68.1 67.0 41.3 37.1 96.4

 

0.66 0.62 0.102 0.219 0.147 0.143   0.49 0.47 2.11 0.80 1.15 0.39

Con vs. T  

0.54 0.54 0.54 0.57 0.25 0.54   0.52 0.60 0.53 0.28 0.79 0.68

L  

0.24 0.23 0.21 0.24 0.10 0.24   0.16 0.18 0.36 0.41 0.54 0.14

Q  

0.94 0.93 0.89 0.90 0.86 0.91   0.91 0.88 0.72 0.89 0.89 0.82

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. n = 32 for all variables (n represents number of observations used in the statistical analysis) except starch (n = 31). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 4 DMI data are for the fecal collection period. 2

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Table 6. Effect of dietary Capsicum oleoresin (CAP) on nitrogen utilization and urinary purine derivatives excretion in dairy cows Treatment1 Item N intake, g/d N excretion or secretion, g/d   Urine N  UUN4, g/d   Fecal N   Total excreta N   Milk N N intake, %   Urine N   Fecal N   Total excreta N   Milk N Urine output, kg/d Urinary PD5 excretion, mmol/d  Allantoin   Uric acid   Total PD

P-value3

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

703   194 147 243 438 245   28.1 34.3 62.4 35.3 16.1   422 69.7 492

707   204 158 245 448 250   28.9 34.3 63.2 35.7 16.8   431 68.3 499

689   197 150 229 426 247   29.8 32.8 62.6 37.3 16.3   407 74.8 482

679   201 158 226 428 240   29.9 33.0 63.0 36.0 16.4   389 64.4 453

16.3   6.7 11.0 16.9 13.2 6.1   1.75 2.11 1.44 0.96 0.33   27.6 2.88 27.8

0.54   0.30 0.13 0.36 0.76 0.85   0.35 0.53 0.77 0.43 0.38   0.63 0.88 0.64

0.21   0.53 0.22 0.14 0.29 0.31   0.30 0.36 0.87 0.58 0.93   0.25 0.29 0.23

0.89   0.69 0.83 0.82 1.00 0.27   0.66 0.72 0.93 0.32 0.55   0.80 0.11 0.67

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. n = 32 for all variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 4 UUN = urinary urea nitrogen. 5 PD = purine derivatives. 2

CAP in the current experiment had no effect on DMI. The CAP product contained fat to reduce its pungent taste, which may have resulted in lack of effect on DMI. The application rate was also somewhat higher in the beef studies compared with the current experiment. For example, the amount of Capsicum oleoresin used in the study of Rodríguez-Prado et al. (2012) was 2.5 to 10.6 mg/kg of DMI, whereas application rate of Capsicum oleoresin was 1.8 to 7.5 mg/kg of DMI in the current

study. Tager and Krause (2011) also reported no effect of Capsicum on DMI in dairy cows. The combination of dietary nutrient supplies and quadratic increases of milk, 4% FCM, and ECM yields with CAP supplementation in the current experiment resulted in negative NEL balances (−0.9 Mcal/d) for both C250 and C500 treatment. The negative energy balance may have led to mobilization of body fat reserves, which may be responsible for the observed qua-

Table 7. Effect of dietary Capsicum oleoresin (CAP) on blood chemistry in dairy cows Treatment1

P-value3

Item

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

BHBA, µM NEFA, mM Insulin, µIU/mL Glucose, mg/dL BUN, mg/dL Creatinine, mg/dL Total protein, g/dL Albumin, g/dL Globulin, g/dL Cholesterol, mg/dL Amylase, U/L P, mg/dL Ca, mg/dL Na, mM K, mM Cl, mM

767 0.14 10.8 62.1 16.1 0.68 7.83 3.08 4.74 282 47.1 6.14 10.1 147 4.38 104

863 0.13 13.5 60.3 17.4 0.69 7.88 3.09 4.78 285 55.0 5.84 10.1 146 4.18 103

948 0.16 10.0 59.6 17.1 0.66 7.90 3.11 4.78 288 48.0 5.91 10.1 149 4.16 104

827 0.13 11.0 61.0 17.3 0.67 7.69 3.02 4.66 272 41.8 5.98 9.94 144 4.01 101

84.9 0.009 1.23 1.94 0.70 0.018 0.107 0.037 0.085 5.6 4.81 0.119 0.101 1.6 0.132 1.32

0.07 0.71 0.57 0.23 0.02 0.59 0.97 0.92 0.96 0.69 0.81 0.09 0.37 0.91 <0.01 0.42

0.49 0.90 0.59 0.65 0.11 0.54 0.21 0.25 0.36 0.24 0.13 0.55 0.13 0.24 <0.01 0.13

0.02 0.55 0.78 0.17 0.17 0.66 0.18 0.21 0.31 0.07 0.18 0.13 0.65 0.11 0.39 0.58

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d CAP. Highest SEM shown; n = 57 for amylase, n = 64 for all other variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 2

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Table 8. Effect of dietary Capsicum oleoresin (CAP) on relative abundance (as percentage1 of total sequences) of rumen major bacterial order in dairy cows Treatment2 Order Clostridiales Bacteroidales Erysipelotrichales Bifidobacteriales Coriobacteriales Fibrobacterales Methanobacteriales Synergistales Chromatiales Spirochaetales Bacillales Desulfovibrionales Sphingobacteriales Planctomycetales Aeromonadales Lactobacillales

P-value4

Control

C250

C500

C1000

SEM3

Con vs. T

L

Q

60.1 27.6 2.41 1.01 1.49 1.17 1.02 0.97 1.33 0.90 0.36 0.20 0.09 0.15 0.10 0.08

60.8 25.0 3.08 1.71 1.27 1.31 0.91 1.22 1.37 1.38 0.35 0.21 0.11 0.10 0.10 0.09

65.9 17.0 3.65 3.76 1.98 1.35 1.56 1.39 0.83 0.92 0.27 0.06 0.16 0.09 0.09 0.11

62.8 21.4 2.95 3.35 2.10 1.47 1.22 1.08 1.02 0.83 0.31 0.09 0.11 0.07 0.08 0.06

2.51 1.73 0.439 1.393 0.560 0.300 0.235 0.070 0.769 0.150 0.090 0.055 0.045 0.046 0.050 0.021

0.17 0.02 0.15 0.12 0.50 0.37 0.54 0.06 0.60 0.20 0.67 0.19 0.57 0.28 0.98 0.78

0.20 0.03 0.41 0.10 0.19 0.32 0.45 0.51 0.51 0.11 0.66 0.10 0.79 0.29 0.83 0.55

0.15 0.03 0.12 0.27 1.00 0.81 0.45 0.03 0.66 0.09 0.69 0.34 0.44 0.63 0.91 0.22

1

The percentage represents the percentage of the total sequences analyzed within the sample. Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. 3 n = 12 for all variables (n represents number of observations used in the statistical analysis). 4 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 2

dratic increase in serum BHBA concentration with the CAP treatments (McArt et al., 2012). This potentially enhanced fat mobilization in the CAP-treated cows

may explain the numerical trend for increased milk fat concentration and the quadratic increase in 4% FCM and ECM, compared with the control cows. This hy-

Table 9. Effect of dietary Capsicum oleoresin (CAP) on relative abundance (as percentage1 of total sequences) of rumen major bacterial genera in dairy cows Treatment2 Genus Ruminococcaceae Prevotella Acetitomaculum Butyrivibrio Prevotellaceae Ruminococcus Roseburia Blautia Saccharofermentans Bifidobacterium Coprococcus Fecalibacterium Syntrophococcus Fibrobacter Synergistaceae Dorea Methanobrevibacter Erysipelotrichaceae Thioalkalibacter Olsenella Treponema

P-value4

Control

C250

C500

C1000

SEM3

Con vs. T

L

Q

21.4 18.8 6.45 5.07 6.12 3.99 4.58 3.28 2.62 0.66 1.56 1.48 1.60 1.17 0.95 0.89 0.89 0.85 1.22 1.00 0.90

22.2 16.4 6.51 6.72 6.09 3.36 2.48 3.41 3.52 1.47 1.89 1.74 1.19 1.31 1.20 1.17 0.82 0.96 1.27 0.62 1.37

22.4 11.3 9.98 6.97 3.77 3.85 2.73 3.10 2.34 3.14 2.01 1.96 1.34 1.35 1.38 1.41 1.45 1.18 0.77 1.16 0.92

22.6 15.0 6.89 6.20 4.35 3.29 3.49 3.15 2.99 2.70 1.74 1.42 2.06 1.47 1.06 1.00 1.12 1.25 0.96 1.40 0.83

2.83 1.28 2.793 1.080 0.834 0.352 0.590 0.204 0.536 1.249 0.369 0.134 0.508 0.250 0.070 0.123 0.223 0.284 0.716 0.443 0.149

0.74 0.04 0.68 <0.01 0.17 0.30 0.04 0.68 0.65 0.15 0.45 0.21 0.80 0.37 0.05 0.10 0.46 0.13 0.61 0.87 0.21

0.77 0.08 0.82 0.03 0.11 0.34 0.38 0.28 0.96 0.15 0.79 0.59 0.13 0.32 0.48 0.64 0.40 0.08 0.52 0.28 0.11

0.87 0.04 0.47 <0.01 0.32 0.98 0.05 0.85 0.94 0.28 0.39 0.04 0.10 0.81 0.02 0.04 0.40 0.49 0.65 0.61 0.09

1

The percentage represents the percentage of the total sequences analyzed within the sample. Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. 3 n = 12 for all variables (n represents number of observations used in the statistical analysis). 4 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 2

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Table 10. Effect of dietary Capsicum oleoresin (CAP) on T cell phenotypes and phagocytosis of neutrophils in dairy cows Treatment1 Item

Control

T cell phenotypes, %  CD4+CD25 CD4-CD25+  CD4+CD25+   Total CD4   Total CD25  CD8+γδ CD8-γδ+  CD8+γδ+   Total CD8   Total γδ Neutrophil phagocytosis   Positive cells, %  MFI4

 

9.50 8.00 10.8 20.3 18.8 6.08 8.62 2.69 8.77 11.3   77.8 143

C250  

8.03 8.41 12.5 20.5 20.9 4.97 9.34 2.25 7.22 11.6   81.2 177

P-value3

C500  

6.98 8.40 11.9 18.9 20.3 5.81 9.38 2.36 8.17 11.7   82.3 181

SEM2

C1000  

7.83 7.91 11.3 19.2 19.2 5.40 8.73 2.18 7.57 10.9   75.9 140

 

1.297 0.757 1.32 1.24 1.95 0.801 0.589 0.258 0.856 0.70   4.09 19.9

Con vs. T  

0.15 0.75 0.19 0.64 0.31 0.41 0.49 0.16 0.29 0.91   0.48 0.31

L  

0.32 0.83 0.93 0.46 0.95 0.72 0.95 0.25 0.53 0.64   0.78 0.84

Q  

0.21 0.51 0.15 0.79 0.20 0.72 0.31 0.57 0.65 0.47   0.20 0.08

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. n = 32 for all variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 4 MFI = mean fluorescence intensity, arbitrary units. 2

pothesis, however, was not supported by the similar serum NEFA concentrations between the CAP-treated and control cows. Milk fat concentration was, on average, 3.03% in our experiment, which is lower than typical values for Holstein cows (CDCB, 2013) and is indicative of milk fat depression. This may have been caused by the high starch content of the corn silage (41.4%) and high proportion of corn silage in the basal diet. Diets rich in starch are known to cause milk fat depression by de-

creasing mammary lipogenesis (Shingfield et al., 2010) and an increase of milk fat 18:1 trans-10 has been consistently associated with milk fat depression (Shingfield and Griinari, 2007). Concentrations of milk fat 18:1 trans-10 in the current study were 2.19 to 2.53% for all treatment, which could cause 20 to 22% reduction in milk fat in lactating cows according to Shingfield et al. (2010). The relatively low rumen pH for all treatments is also reflective of high level of starch fermentation in the rumen. A decrease by CAP in cis-9,trans-11 CLA

Table 11. Effect of dietary Capsicum oleoresin (CAP) on blood cell counts in dairy cows Treatment1 Item 3

White blood cells, 10 /μL  Neutrophils  Lymphocytes  Monocytes  Eosinophils  Basophils Total, %  Neutrophils  Lymphocytes  Monocytes  Eosinophils  Basophils  Neutrophil:lymphocyte   Red blood cells, 106/μL   Hemoglobin, g/dL   Hematocrit, %   Platelets, 103/μL   Mean platelet volume, fL

P-value3

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

7.06 3.59 2.99 0.22 0.25 0.01   51.1 42.3 3.07 3.41 0.15 1.26 5.66 8.59 24.7 405 6.21

7.29 3.75 3.00 0.21 0.31 0.02   51.1 41.6 2.81 4.25 0.23 1.28 5.70 8.84 24.9 371 6.20

7.06 3.62 2.91 0.23 0.29 0.01   51.7 41.0 3.16 4.02 0.17 1.29 5.85 8.96 25.5 380 6.18

8.01 4.56 2.85 0.22 0.37 0.02   55.1 37.0 2.82 4.78 0.24 1.73 5.66 8.77 24.7 352 6.14

0.298 0.252 0.076 0.019 0.038 0.005   1.31 1.20 0.20 0.496 0.048 0.084 0.254 0.106 0.67 48.9 0.193

0.29 0.19 0.47 0.92 0.02 0.27   0.14 0.04 0.54 0.03 0.22 0.06 0.30 <0.01 0.36 0.05 0.76

0.04 0.01 0.16 0.94 0.01 0.30   <0.01 <0.01 0.57 0.04 0.27 <0.01 0.90 0.13 0.97 0.04 0.59

0.36 0.33 0.89 0.68 0.46 0.99   0.43 0.30 0.72 0.34 0.96 0.24 0.04 <0.01 0.07 0.72 0.94

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. n = 64 for all variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 2

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CAPSICUM OLEORESIN IN DAIRY COWS

Table 12. Effect of dietary Capsicum oleoresin (CAP) on oxidative stress markers in dairy cows Treatment1

P-value3

Item

Control

C250

C500

C1000

SEM2

Con vs. T

L

Q

8-Isoprostane, pg/mL TBARS,4 µM ORAC,5 µM

16.5 3.11 8,967

14.2 3.40 9,336

15.3 3.30 9,411

14.6 4.29 9,414

3.72 0.64 322.0

0.25 0.31 0.31

0.44 0.45 0.42

0.99 0.69 0.52

1

Control = 0 mg/d of CAP; C250 = 250 mg/d of CAP; C500 = 500 mg/d of CAP; C1000 = 1,000 mg/d of CAP. n = 64 for all variables (n represents number of observations used in the statistical analysis). 3 Con vs. T = control vs. treatment; L = linear effect of CAP; Q = quadratic effect of CAP. 4 TBARS = thiobarbituric acid reactive substances; trolox equivalents. 5 ORAC = oxygen radical absorbance capacity. 2

is consistent with the results for 18:1 trans-11 in the current experiment because cis-9,trans-11 CLA is synthesized in the mammary gland from 18:1 trans-11 by Δ9-desaturase (Griinari et al., 2000). Overall, rumen fermentation parameters were not affected by CAP in the current experiment, although a slight decrease (P = 0.06) in pH was observed. These results are consistent with those reported by Tager and Krause (2011), in which ruminal ammonia and VFA concentrations were not affected by 250 mg/d of a Capsicum product in lactating dairy cows. Others, however, have reported decreased pH and acetate concentration and increased ammonia concentration in the rumen of beef cattle supplemented with encapsulated Capsicum oleoresin (Rodríguez-Prado et al., 2012). Explanation of the increased BUN concentration by CAP in the current experiment remains uncertain because ruminal ammonia concentration and MUN were not different from the control. None of the other blood chemistry variables analyzed, with the exception of BHBA, were affected by CAP. The BUN results were not consistent with previous data from experiments with nonruminant species. For example, capsaicin or Capsicum extract were reported to decrease BUN concentration in rats (Monsereenusorn, 1983; Jung et al., 2014). Antimicrobial properties of capsaicin or Capsicum oleoresin against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, Helicobacter pylori, Botrytis cinerea, and Aspergillus niger were reported in vitro (al-Delaimy and Ali, 1970; Cichewicz and Thorpe, 1996; Jones et al., 1997; Xing et al., 2006). Therefore, some antimicrobial effects of Capsicum in the rumen were expected in the current experiment. Supplementation of CAP decreased Prevotella species in the rumen, gram-negative bacteria that degrade proteins and are involved in the uptake and fermentation of peptides (Stewart et al., 1997). Ruminal ammonia concentration, however, was not affected by CAP in the current experiment. Butyrivibrio species are associated

with biohydrogenation of FA in the rumen (Jenkins et al., 2008). It is known that milk fat 18:1 trans-11 and cis-9,trans-11 CLA are formed from linoleic acid by Butyrivibrio fibrisolvens. The observed quadratic increase in the abundance of Butyrivibrio species and B. fibrisolvens (P = 0.08; 0.01, 0.03, 0.03, and 0.02% for control, C250, C500, and C1000, respectively; data not shown in tables) is consistent with the decrease in PUFA, but did not result in increased milk fat 18:1 trans-11 and cis-9,trans-11 CLA. Capsaicinoids have been shown to have immunomodulatory effects in vitro and in vivo in rats (Sancho et al., 2002; Park et al., 2004; Liu et al., 2012). Capsaicin and Capsicum oleoresin reduced IL-2 and IFN-γ production in murine Peyer’s patches, lymphoid tissues where systemic immune responses are induced in the intestine (Takano et al., 2007). Takano et al. (2007) also reported that capsaicin and Capsicum oleoresin decreased proportion of CD4+ cells in Peyer’s patches. These anti-inflammatory effects of capsaicin were shown by Park et al. (2004), who reported that capsaicin suppressed tumor necrosis factor (TNF) production in murine macrophage. In food animals, Capsicum oleoresin linearly decreased TNF, IL-1B, and transforming growth factor production of LPS-induced porcine macrophages in vitro (Liu et al., 2012), suggesting an anti-inflammatory effect of capsaicin on macrophages in pigs. Oh et al. (2013) infused Capsicum oleoresin (2 g/d) into the abomasum of lactating dairy cows and measured inflammatory cytokines production of LPSinduced peripheral blood mononuclear cells ex vivo and subpopulations of T cells in blood. In that experiment, the abomasal infusion of Capsicum oleoresin did not affect inflammatory cytokines such as IFN-γ, IL-6, and TNF, but increased the proportion of CD4+ cells in blood. In the current experiment, however, we did not observe any effect of dietary CAP on populations of T cells. It is possible that the different doses used in the 2 experiments influenced the T cell response. In addition, the mode of supplementation (abomasal infusion vs. Journal of Dairy Science Vol. 98 No. 9, 2015

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Oh et al.

TMR top-dressing) might have influenced the effects of CAP. Neutrophils are the most abundant type of white blood cell and play critical roles in innate immunity by killing bacteria and inducing an adaptive immune response (Witko-Sarsat et al., 2000). Supplementation of the diet with CAP in the current experiment increased neutrophil counts in blood and neutrophil activity in phagocytosis. Neutrophils may be directly affected by CAP because they have a transient receptor potential cation channel subfamily V member 1 (TRPV1) channel (Heiner et al., 2003), or indirectly affected via release of neuronal peptides (Zimmerman et al., 1992). In the current study, it is possible that neurons activated by CAP facilitated neutrophil function through secretion of neuropeptides. Zhukova and Makarova (2002) indicated that neuropeptides regulated by capsaicin increased count and activity of neutrophils in rats. Franco-Penteado et al. (2006) reported that capsaicin increased neutrophil production in rat bone marrow. An increase of the neutrophil-to-lymphocyte ratio was suggested as an indicator of an acute stress response (Weiss and Wardrop, 2010). In the current experiment, CAP increased the ratio of neutrophils to lymphocytes as well as the counts of neutrophils and eosinophils. Eosinophils are granulocytes and are involved in acute phase responses, such as host protection against parasites (Rothenberg and Hogan, 2006). In our previous study, we reported that abomasal infusion of Capsicum oleoresin for 5 d did not affect 8-isoprostane and oxygen radical absorbance capacity in blood of dairy cows (Oh et al., 2013). This is consistent with data from the current experiment, although CAP was supplied to the cows for longer period (21 d) and through the TMR. In studies with rats, however, capsaicin reduced oxidative stress markers such as thiobarbituric acid reactive substances or malondialdehyde in serum and the animals’ liver, lung, kidney, and muscle (Lee at al., 2003; Manjunatha and Srinivasan, 2006). CONCLUSIONS

Our results suggest that dietary supplementation of CAP has subtle or no effects on feed intake, rumen fermentation, nutrients digestibility, blood chemistry, T cell phenotypes, and antioxidant status in lactating dairy cows. However, CAP appeared to have a positive effect, perhaps through enhanced mobilization of body fat reserves, on the energy balance of the cows, resulting in increased ECM yield. Capsicum facilitated neutrophil activity and immune cells related to the acute phase response. Journal of Dairy Science Vol. 98 No. 9, 2015

ACKNOWLEDGMENTS

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Journal of Dairy Science Vol. 98 No. 9, 2015